Polymeric derivatives of camptothecins

ABSTRACT

The invention relates to polymeric conjugates of 20-O-[glycyl-aminoacyl-glycyl]-camptothecins and a process for producing the same.

This application is a 371 of PCT/EP98/06048 filed Sep. 22, 1998.

The invention relates to polymeric conjugates of20-O-[glycyl-aminoacyl-glycyl]camptothecins. Our WO-95/10304 describesand claims conjugates of camptothecins linked to a polymer through apeptidyl spacer. We have now found that the conjugates in which thespacer is a glycyl-aminoacyl-glycyl are of exceptional value asantitumor agent and are endowed with remarkable antitumor activity andreduced toxicity in comparison with the free drug.

Particularly the present invention provides polymeric conjugates of theformula (1) consisting of:

(i) from 85 to 97 mol % of N-(2-hydroxypropyl)methacryloylamide unitsrepresented by formula (3)

(ii) from 3 to 15 mol % of20-O-(N-methacryloyl-glycyl-aminoacyl-glycyl)-camptothecin unitsrepresented by formula (4).

 wherein n is from 2 to 8, —[O-CPT] represents the residue of acamptothecin of formula (2)

which is linked at the C-20 position and in which each of R₁, R₂, R₃, R₄and R₅ which are the same or different, is hydrogen, C₁-C₁₂ linear orbranched alkyl, nitro, amino, (CH₂)_(a)NR₆R₇ in which a is from 0 to 4and R₆ and R₇ are hydrogen or one of R₆ or R₇ is hydrogen and the otherof R₆ or R₇ is C₁-C₆ alkyl, or NR₆R₇ represents a piperazino orN-alkyl-piperazino ring optionally substituted with C₁-C₆ linear orbranched alkyl or piperidino ring, (CH₂)_(a)NHCOR₈ in which a is asabove defined and R₈ is C₁-C₈ linear or branched alkyl or a group NR₆R₇as above, hydroxy or O-CO-R₈ in which R₈ is as above defined orrepresents a 1-piperidino ring or 1,4′-bipiperidine, or R₂ and R₃ takentogether represent the residue O—(CH₂)_(b)—O, in which b is 1 or 2, orR₄ and R₅ represent the residue (CH₂)_(m), in which m is from 2 to 4, orthe residue CH₂—O—CH₂ or CH₂NHCH₂

and (iii) from 0 to 12 mol % of N-methacryloyl-glycine orN-(2-hydroxy-propyl)methacryloyl-glycinamide units represented byformula (5)

 wherein [Z] represents a hydroxy group or a residue of formula—NH—CH₂—CH(OH)—CH₃.

The polymeric conjugates of the formula (1) may be indicated asMAG-CPT(s) and may also be represented as follows:

[(3)]_(x); [(4)]_(y); [(5)]_(z) wherein (3), (4) and (5) are units ofthe formula as above defined, and x is from 85 to 97 mol %, y is from 3to 15 mol % and z is from 0 to 12 mol %.

A preferred embodiment of compounds of the present invention are thosein which —[O-CPT] in formula (4) is a residue of a camptothecin offormula (2) selected from:

camptothecin [2a: R₁=R₂=R₃=R₄=R₅=H];

9-aminocamptothecin [2b: R₁=R₂=R₃=R₅=H, R₄=NH₂];

9-nitrocamptothecin [2c: R₁=R₂=R₃=R₅=H, R₄=NO₂];

7-ethyl-10-hydroxycamptothecin [2d: R₁=R₂=R₄=H, R₃=OH, R₅=CH₂CH₃];

7-ethyl-10-[1,4′-bipiperidinyl]carbonyloxycamptothecin [2e: R₁=R₂=R₄=H,R₃=OCO-[1,4′-bipiperidinyl], R₅=CH₂CH₃],

7-methylendimethylamino-10-hydroxycamptothecin [2f: R₁=R₂=R₄=H, R₃=OH,R₅=CH₂N(CH₃)₂] and

7-[methylen-(4′-methylpiperazino)]-9,10-ethylendioxycamptothecin [2g:R₁=R₄=H, R₂,R₃=O—CH₂CH₂—O, R₅=methylen-(4′-methylpiperazino)].

Preferably, the polymeric conjugates of the formula (1) contain theN-(2-hydroxypropyl) methacryloyl amide units represented by the formula(3) in a proportion of 90% or more; more preferably 90%. The conjugatemay also contain from 3 to 10 mol % of the units represented by theformula (4), more preferably 10 mol % of such units. Preferably theconjugate of formula (1) does not contain residues of formula (5), i.e.z is 0. Content of active camptothecin derivative of formula (2) in theconjugate of formula (1) may be from 2 to 15% (weight/weight), morepreferably 10% (w/w). The preparation of the compounds of the presentinvention may be carried out by a process (herein named Route I) whichcomprises reacting 20-O-(aminoacyl-glycyl) camptothecin derivative offormula (6)

NH₂—(CH₂)_(n)—CO-Gly-[OCPT]  (6)

wherein n and [OCPT] are as above defined, with a polymer (B) consistingessentially of:

from 85 to 97 mol % of N-(2-hydroxypropyl)methacryloylamide unitsrepresented by formula (3) as above defined,

and from 3 to 15 mol % of N-methacryloyl-glycyl derivative unitsrepresented by formula (7)

wherein [Y] is the residue of an active ester, preferably p-nitrophenylester, or a hydroxy group; and optionally displacing the remainingactive ester groups with 1-amino-2-propanol. The condensation ofderivative of formula (6) with the polymer of formula (B), is carriedout in conditions capable of preserving the nature of linkage betweencamptothecin and the aminoacyl-glycyl spacer as well as that of theconjugate.

Polymers of formula (B), consisting ofN-(2-hydroxypropyl)-methacryloylamide units of formula (3) and ofN-methacryloyl-glycine units of formula (7), are prepared bycopolymerization of N-(2-hydroxypropyl)methacrylamide withN-methacryloyl-glycine or N-methacryloyl-glycine active-esterderivatives, as described in Makromol.Chem. 178, 2159 (1977). Theresidue [Y] may represent a phenoxy group which is substituted on thephenyl ring by one ore more electron-withdrawing groups, such as nitroor halogen. Preferably the residue [Y] represents p-nitro phenoxy.

Reaction between (6) and (B) to form polymeric-drug-conjugate of formula(1) of the present invention can typically carried out at temperaturefrom 15 to 30° C., preferably at room temperature for 15 hours; then theaminolysis of the remaining active ester groups can be performed in thepresence of 1-amino-2-propanol at room temperature, from 0.5 to 1 hour.The conjugate suitably is precipitate with ethyl acetate dissolved inethanol and reprecipitated with ethyl acetate.

For example, the polymer (B) in which [Y] represents the residue of anactive ester, provided at a concentration of 15% (weight/volume) in drydimethylsulfoxide, is treated with 20-O-(aminoacyl-glycyl)camptothecinderivative (6), 3% (w/v), at room temperature for 15 hours. Then1-amino-2-propanol, 0.1% (w/v) is added and the reaction mixture is keptat room temperature for 1 hour. The polymeric-drug-conjugate. MAG-CPTs,can be precipitated with ethyl acetate, collected, washed with ethylacetate, then dissolved with absolute ethanol at a concentration of 10%(weight/volume) and precipitated again with ethyl acetate to give theconjugates of formula (1) according to the invention. The content ofcamptothecin in the polymeric conjugate of the invention is determinedby HPLC or absorbance spectroscopy analysis.

The compounds of the formula (1) c an be also prepared by a process(herein named Route II) which comprises the polymerization betweenN-(2-hydroxypropyl) methacrylamide of the formula (8)

and 20-O-[methacryloyl-glycyl-(aminoacyl )-glycyl]camptothecinderivatives of the formula (9)

wherein n and [OCPT] are as above defined, in conditions capable ofpreserving the nature of linkage between camptothecin and spacerglycyl-aminoacyl-glycyl as well as that of the conjugate.

Reaction between (8) and (9) can typically carried out at temperaturefrom 50 to 70° C., preferably at 60° C., from 6 to 24 hours, preferablyfor 15 hours, in aprotic solvent such as dimethylsulfoxide and inpresence of catalyst, such as 2,2′-azobisisobutyronitrile. The conjugateis precipitate with ethyl acetate dissolved in ethanol andreprecipitated with ethyl acetate.

For example N-(2-hydroxypropyl)methacrylamide (8), provided at aconcentration of 22% (w/v) and20-O-[methacryloyl-glycyl-(6-aminohexanoyl)-glycyl]camptothecinderivative (9) at concentration of 6% (w/v) in dry dimethylsulfoxide areheated at 60° C. under nitrogen and then added with2,2′-azobisisobutyronitrile at concentration of 1.3% (w/v). The mixtureis kept under stirring for 24 hours. After that, the reaction mixture iscooled at room temperature and the conjugate suitably is precipitatewith ethyl acetate, dissolved in ethanol and reprecipitated with ethylacetate to give conjugate of formula (1) according to the invention.

The invention also provides 20-O-acylamino-glycyl-camptothecinderivatives (6) as defined above and their salt derivatives.

The present invention further provides a process for preparing20-O-(aminoacyl-glycyl) camptothecin derivatives (6), which processcomprises condensing the residue of the formula (2) as defined abovewith a N-protected aminoacyl-glycyl derivative of formula (10):

R₉—HN—(CH₂)_(n)—CO-Gly-[P]  (10)

wherein n is as above defined, R₉ represents an amino-protecting groupsuch as Boc, FMOC, triphenylsilyl, diphenylmethylene or triphenylmethyl,and [P] is a residue of an activated ester, such as p-nitro phenoxy orN-hydroxysuccinimido to give a compound represented by formula (11):

R₉—NH—(CH₂)_(n)—CO-Gly-[OCPT]  (11)

wherein n, [OCPT] and R₉ are as defined above; and removing theN-protecting group from the resulting compound.

Preparation of compounds of formula (10) follows standard syntheticprocedures that are known from the literature. SuitableN-protected-aminoacyl derivatives of formula (10) include:6-N-(triphenylmethyl)hexanonyl glycyl p-nitrophenylester (10a),6-N-(tert-Butoxy carbonyl)hexanonyl-glycyl p-nitrophenyl ester (10b).

Thus, for example, camptothecin (2a) may be allowed to react with amolar excess, for example up to five-fold molar excess or more,especially 2 mol.equivalent, of a N-protected-aminoacyl derivative offormula 10 in anhydrous solvent such as dry dimethylsulfoxide in thepresence of 4-dimethylaminopyridine.

The reaction can typically be effected for from 8 to 48 hours. Thereaction is typically carried out at a temperature from 15 to 40° C. Thetemporary amino-protected group R₉ is removed by an appropriatedeprotecting agent to give the 20-O-(aminoacyl-glycyl)camptothecin offormula (6a). Deprotcction may be therefore achieved by acid treatment,such as treatment with 1.5N aqueous hydrochloric acid in acetic acid or90% aqueous trifluoroacetic acid from one to 6 hours at temperature from10 to 30° C.; preferably for two hours at room temperature.

The invention also provides20-O-[methacryloyl-glycyl-(aminoacyl)-glycyl]camptothecin derivatives(9) as above defined and a process for their preparation, whichcomprises condensing camptothecin derivatives of formula (6) as abovedefined with N-methacryloyl-glycyl of formula (7′),

wherein [Y′] is a leaving group. Thus, for example,20-O-[aminoacyl-glycyl]camptothecin (6a), provided at a concentration of25% (w/v) in dry dimethyl sulfoxide, is reacted with ofN-(methacryloyl-glycyl) p-nitrophenyl ester ((7′), [Y′]=p-nitro-phenol),13% (w/v) in presence of equivalent amount of base, such astriethylamine, for 15 at room temperature. The final derivative isisolated by precipitation and purified by chromatography. The compoundsof the formula (8) and the polymer (B) are known or may be prepared byknown synthetic methods.

All the camptothecin derivatives of formula (2) are known, see forexample Medicinal Research Reviews, Vol 17, No.4, 367-425, 1997, or maybe prepared by means of well known perocedures.

The polymer-bound conjugates of formula (1) are in the range of 5,000 to45,000 molecular weight, preferably from 8,000 to 35,000. Polymeric drugconjugates of formula (1) are water soluble and show remarkableantitumor activity and reduced toxicity in comparison with the freecamptothecin.

Antitumor Activity

Campound Al was tested on human colon carcinoma (HT29) transplanted innude mice, in comparison with the free drug (2a) by i.v. route. A1 wasfound non toxic and gave >95% tumor inhibition at all tested doses withan exceptional high number of tumor free animals at the end of theexperiment (90 days), Table 1. Also compound A2, tested on the samemodel in comparison with free 7-ethyl-10-hydroxycamptothecin (2d), wasfound active and not toxic and gave 98% tumor inhibition at the highesttested dose of 40 mg/kg (Table 2). Compound A1 was also tested by i.v.route on a broad panel of other human tumor models: A2780 ovary ca., MX1mammary ca., A549 NSC lung ca. and M14 melanoma. Compound A1 was moreactive respect to the corresponding free camptothecin (2a) and gave alarge number of cured animals.

Activity against MX1 mammary carcinoma, A2780 ovary carcinoma and M14melanoma, in comparison with free camptothecin, are reportedrespectively in Table 3, 4 and 5. Compound A1 was very active againstthese tumor models for which total inhibition of tumor growth wasobserved with total 7/7 and 8/8 cured mice upon repeated i.v.administration of the drug either at 15 or 20 mg/kg at the schedule ofq4dx6. Compound A1 was found also active against NSC lung carcinoma at20 mg/kg (iv q4dx6) with TI % 94 and with remarkable tumor growth dalayof 70 days never observed with other useful chemotherapeutic agents(Table 6).

TABLE 1 Antitumor Activity of A1 on human colon carcinoma (HT29) incomparison with (2a). Treatment iv q4dx6. ΔTGD Dose Total Dose TI %Tumor (0.5 g) Compound mg/kg mg/kg 37^(th) day Tox Free days A1 15 90 970/7 2/7 >88 17.5 105 98 0/7 1/7 >88 20 120 99 0/7 5/7 >88 22.5 135 990/7 6/7 >88 2a 12.5 75 94 0/7 0/7 43

Tumor fragment were implanted sc. Treatment started when tumor waspalpable.

TI % (tumor inhibition %) was calculated at day 37.

ΔTGD: Tumor growth delay of treated animals—Tumor growth delay ofcontrol animals.

TABLE 2 Antitumor Activity of A2 on human colon carcinoma (HT29) incomparison with (2d). Total ΔTGD Treatment Dose Dose TI % (0.5 g)Compound schedule mg/kg mg/kg 37^(th) day Tox days A2 iv q4dx8 12 80 780/7 0 iv q4dx8 20 160 91 0/7 21 iv q4dx8 40 320 98 0/7 56 2d iv q4dx6 20120 97 1/7 50

Tumor fragments were implanted sc. Treatment started when tumor waspalpable.

TI % (tumor inhibition %) was calculated at day 37.

ΔTGD: Tumor growth delay of treated animals—Tumor growth delay ofcontrol animals.

TABLE 3 Antitumor activity of A1 against MX1, human mammary ca., incomparison with camptothecin (2a). dose/ total ΔTGD treatment dose TI(0.5 g) Tumor Compound schedule mg/kg % Tox days free A1 iv q4dx6 20/120100 0/8 >88 8/8 2a iv q4dx4 10/40  100 3/8 69 0/8

Tumor fragments were implanted sc., treatment started when tumors werepalpable.

Doses are expressed as camptothecin equivalents.

TI %: Inhibition of tumor growth 1 week after the last treatment

Tox: Number mice died for toxicity/total number mice

ΔTGD: Tumor growth delay treated. Tumor growth delay control

Tumor Free: cured mice at day 90 after tumor implant

TABLE 4 Antitumor activity of A1 against A2780, human ovaric ca., incomparison with camptothecin (2a). dose/ total ΔTGD treatment dose TI(0.5 g) Tumor Compound schedule mg/kg % Tox days free A1 iv q4dx6 15/90 100 0/7 >82 7/7 iv q4dx6 20/120 100 0/7 >82 7/7 2A iv q4dx3 10/30  nd7/7 nd 0/7

Tumor fragments were implanted sc., treatment started when tumors werepalpable.

Doses are expressed as camptothecin equivalents.

TI %: Inhibition of tumor growth 1 week after the last treatment

Tox: Number mice died for toxicity/total number mice

ΔTGD: Tumor growth delay treated. Tumor growth delay control

Tumor Free: cured mice at day 90 after tumor implant

TABLE 5 Antitumor activity of A1 against M14, human melanoma., incomparison with camptothecin (2a). dose/ total ΔTGD treatment dose TI(0.5 g) Tumor Compound schedule mg/kg % Tox days free A1 iv q4dx6 15/90100 0/7 >78 7/7 iv q4dx6 20/120 100 0/7 >78 7/7 2a iv q4dx6 10/60 94 0/734 0/7

Tumor fragments were implanted sc., treatment started when tumors werepalpable.

Doses are expressed as camptothecin equivalents.

TI %: Inhibition of tumor growth 1 week after the last treatment

Tox: Number mice died for toxicity/total number mice

ΔTGD: Tumor growth delay treated. Tumor growth delay control

Tumor Free: cured mice at day 90 after tumor implant

TABLE 6 Antitumor activity of A1 against A549, hunian NSC lung ca., incomparison with camptothecin (2a). dose/ total ΔTGD treatment dose TI(0.5 g) Tumor Compound schedule mg/kg % Tox days free A1 iv q4dx6 15/90 92 0/7 >48 0/7 iv q4dx6 20/120 94 0/7 >70 0/7 2a iv q4dx6 10/60  89 0/74 0/7

Tumor fragments were implanted sc., treatment started when tumors werepalpable.

Doses are expressed as camptothecin equivalents.

TI %: Inhibition of tumor growth 1 week after the last treatment

Tox: Number mice died for toxicity/total number mice

ΔTGD: Tumor growth delay treated. Tumor growth delay control

Tumor Free: cured mice at day 90 after tumor implant

Therefore, the compounds of the present invention are useful in thetreatment of leukemia and solid tumors, such as colon, colo-rectal,gastric, ovarian, mammary, prostate. lung, kidney and also melanomatumors. A human can therefore be treated by a method comprisingadministering thereto a therapeutically effective amount of a polymericconjugate of the invention. The condition of the human patient can thusbe improved. The dosage range adopted will depend on the route ofadministration and on the age, weight and condition of the patient beingtreated. The polymeric conjugates of formula (1) are typicallyadministered by parenteral route, for example intramuscularly,intravenously or by bolus infusion. A suitable dose range is from 1 to1000 mg of camptothecin equivalent per m² body surface area, forinstance from 10 to 100 mg/m².

The polymeric conjugate (1) may be formulated into a pharmaceuticalcomposition together with a pharmaceutically carrier or diluent.Typically they are formulated for parenteral administration, for exampleby dissolution in water for injection or physiological saline.

The following Examples illustrate the invention.

EXAMPLE 1 Preparation of:N-(tert-butyloxycarbonyl)-6-aminohexanoyl-glycyl p-nitrophenyl-ester

[10a: n=5, R₉=Boc, P=p-nitrophenol]

Glycine ethyl ester hydrochloride (9.55g, 68.4 mmol), dissolved withdimethylformamide (100 ml), was added with triethylamine (9.5 ml, 68.4mmol) and then with N-(tert-butyloxy carbonyl)-6-aminohexanoylp-nitrophenyl-ester (19 g, 54 mmol), prepared following the sameprocedure described in our E.P. N°0673258. The reaction mixture was keptfor two hours at room temperature, then the solvent was removed underreduced pressure. The residue was dissolved with ethyl acetate (300 ml)and washed in sequence with cold 1N aqueous hydrochloric acid (3×200ml), water (100 ml), 5% aqueous solution of sodium hydrogen carbonate(2×200 ml) and water (2×200 ml). The organic phase was dried overanhydrous sodium sulphate, then the solvent was removed under reducedpressure. The residue was crystallized from ethyl ether to giveN-(tert-butyloxycarbonyl)-6-aminohexanoyl-glycyl ethyl ester (15 g; TLCon Kieselgel plate F₂₅₄ (Merck), eluting system ethyl ether, R_(f)=0.3)which was suspended with ethanol (150ml) and treated under stirring with1N aqueous sodium hydroxide (48 ml, 48 mmol). After one hour thereaction was added with 1N aqueous hydrochloric acid (48 ml 48 mmol) anddistilled under reduced pressure. The residue was suspended with drytctrahydrofurane (200 ml), added with p-nitrophenol (6,53g, 47 mmol),cooled at 0° C. and then added with a solution of1,3-dicyclohexyl-carbodiimide (9.7 g, 47 mmol) in tetrahydrofurane (100ml). The reaction was left to stand in the same conditions overnight,then filtered on a sintered glass funnel. The solvent was removed underreduced pressure. The residue was crystallized from ethyl ether to give17.5 g of the title compound (10a). TLC on Kieselgel plate F₂₅₄ (Merck),eluting system methylene chloride/methanol (95/5 v/v) R_(f)=0.34.

¹H-NMR (200 MHz, DMSO) δ: 1.34 (s, 9H, t-Bu); 1.0-1.7 [m, 6H, NH—CH₂—(CH₂)₃—CH₂ 13 CO]; 2.15 (t, J=7.2 Hz, 2H, NH—(CH₂)₄—CO); 2.85 [q, J=6.5 Hz,2H, NH—CH ₂—(CH₂)₄—CO]; 4.11 (d, J=5.5 Hz, 2H, CONHCH ₂COO); 6.70 [bs,1H, NH—(CH₂)₅—CO]; 7.40 (m, 2H, aromatic 2,6-H); 8.30 (m, 2H, aromatic3,5-H); 8.43 (t, J=5.5 Hz, 1H. CONHCH₂COO).

EXAMPLE 2 Preparation of:20-O-[(N-tert-butyloxycarbonyl)-6-aminohexanoyl-glycyl]camptothecin

[11a: n=5, R₉=t-Boc, OCPT=(2a)]

Camptothecin (2a; 3.4 g, 10 mmol), suspended with dimethylsulfoxide (50ml), was treated with N-(tert-butyloxycarbonyl)-6-aminohexanoyl-glycylp-nitrophenyl-ester (10a; 6.3 g, 15 mmol) and 4-dimethylaminopyridine(2.4 g, 20 mmol). The reaction mixture was left to stand for 24 hoursand then an addition aliquot of N-(tert-butyloxycarbonyl)-6-aminohexanoyl-glycyl p-nitrophenyl-ester (6.3 g, 15 mmol) was added. After 48hours the reaction mixture was diluted with methylene chloride (500 ml)and washed with 0.2N aqueous hydrochloric acid (2×250 ml) and water(2×250 ml). The organic phase was dried over anhydrous sodium sulphate,then the solvent was removed under reduced pressure. The residue wasdissolved with methylene chloride (100 ml), added with ethyl ether (500ml) and kept at 0° C. overnight to give 5 g of the title compound (11a)in the solid form. TLC on Kieselgel plate F₂₅₄ (Merck), eluting systemmethylene chloride/methanol (95/5 v/v) R_(f)=0.44.

EXAMPLE 3 Preparation of: 20-O-(6-aminohexanoyl-glycyl)camptothecin

[6a: n=5, OCPT=(2a)]

20-O-[(N-tert-butyloxycarbonyl)-6-aminohexanoyl-glycyl]camptothecin(11a, 5 g) was treated with 90% aqueous trifluoroacetic acid (40 ml) forone hour, then the solvent was removed under reduced pressure. Theresidue was triturated with ethyl ether 300 ml) and filtered off. Thesolid was dissolved in methanol (200 ml), reduced to small volume (50ml) under reduced pressure, added with ethyl ether (300 ml). Theprecipitate was collected to give 3.9 g of the title compound (6a).

TLC on Kieselgel plate F₂₅₄ (Merck), eluting system methylenechloride/methanol/acetic acid/water (80/20/7/3 v/v), R_(f)=0.83.

EXAMPLE 4 Preparation of:20-O-[methacryloyl-glycyl-(6-aminohexanoyl-glycyl]camptothecin

[9a: n=5, OCPT=(2a)]

20-O-(6-aminohexanoyl-glycyl)camptothecin trifluoroacetate (6a; 2.53 g,4mmol) was dissolved with anhydrous dimethylsulfoxide (10 ml) and addedwith methacryloyl-glycyl p-nitrophenyl ester (1.32 g, 5 mmol), preparedas described in Makromol.Chem. 178, 2159 (1977), and triethylamine (0.56ml, 4 mmol). After standing overnight at room temperature, the solutionwas poured into water (100 ml) and the precipitate was collected andwashed with water (2×100 ml). The solid material was flashchromatographed on silica gel using as eluting system a mixture ofmethylene chloride/ethanol (95/5 v/v) to give 2.2 g of the titlecompound (9a). TLC on Kieselgel plate F₂₅₄ (Merck), eluting systemmethylene chloride/methanol (9/1 v/v) R_(f)=0.62.

¹H-NMR (200 MHz, DMSO) δ: 0.90 (t, J=7.3 Hz, 3H, CH ₃-18); 1.1-1.6 [m,6H, NHCH₂—(CH₂)₃—CH₂CO]; 2.0-2.2 [m, 4H, CH ₂-19+NH—(CH₂)₄—CH₂—CO]; 2.95(q, J=6.3 Hz, 2H, NH—CH ₂—(CH₂)₄CO): 3.63 (d, J=5.8 Hz, 2H,CONH—CH2-CONH); 3.97 (dd, J=17.8, 5.9 Hz, 1H, CONHCH _(a)CH_(b)COO);4.15 (dd, J=17.8, 5.9 Hz, 1H, CONHCH_(a) CH _(b)COO), 5.29 (s. 2H, CH₂-5); 5.33 [q, J=1.6 Hz, 1H, CH₃CH═C(H _(a))(H_(b))]; 5.48 (s, 2H, CH₂-17); 5.69 [m, 1H, CH₃CH═C(Ha)(H_(b))]; 7.15 (s, 1H, H-14); 7.6-7.9 (m,3H, H-10+H—11+NH—(CH₂₎ ₅CO]; 8.0 (t, J=5.8 Hz, 1H, CO—NH—CH₂CONH); 8.15(m, 2H, H-9+H-12); 8.32 (t, J=5.9 Hz, 1H, CO—NH—CH_(a)CH_(b)COO); 8.69(s, 1H, H-7).

EXAMPLE 5

Preparation of:7-ethyl-10-hydroxy-20-O-(6-aminohexanoyl-glycyl)camptothecin trifluoro

acetate [6b: n=5, OCPT=(2d)]

7-ethyl-10-hydroxy-camptothecin (2d, 0.8 g, 2mmol).N-(tert-butoxycarbonyl)-6-amino-hexanonyl-glycyl p-nitrophenylester(10a; 2.5g, 6 mmol) and 4-dimethylaminopyridine were dissolved with drydimethylsulfoxide (30 ml) and kept at room temperature for 3 days understirring. After that the reaction mixture was poured in 0.1N aqueoushydrochloric acid (500 ml) to give a precipitate which was collected,then dissolved in methylene chloride (300 ml) and washed with water(2×100 ml). The organic phase was separated, dried over anhydrous sodiumsulfate and evaporated under reduced pressure. The residue was treatedwith 90% aqueous trifluoroacetic acid (40 ml) for three hours then thesolvent was removed under reduced pressure and the residue was flashchromatographed on silica gel using a mixture of methylenechloride/acetic acid/methanol (100/5/20 v/v) as eluting system.Fractions containing the title compound were pooled and evaporated underreduced pressure to give 1.02 g of (6b) as trifluoroacetate saltderivative. TLC on Kieselgel plate F₂₅₄ (Merck), eluting systemmethylene chloride/methanol/acetic acid/water (80/20/7/3 v/v),R_(f)=0.4.

¹H-NMR (200 MHz, DMSO) δ: 0.89 (t, J=7.2 Hz, 3H, CH ₃—CH₂—20); 1.1-1.5(m, 9H, NH₂—CH₂—CH ₂—CH ₂—CH ₂—CH₂+CH ₃—CH₂—7); 1.80 (s, 3H, CH ₃—COOH);2.10 (m, 4H, CH₃—CH ₂—20+CH ₂—CONH); 2.53 (t, 6.8 Hz, 2H, CH ₂—NH₂);3.06 (m, 2H, CH₃—CH ₂—7); 3.98, 4.13 (two-dd, J=17.6, 5.7 Hz, 2H,CONH—CH ₂—CO); 5.27 (s, 2H, CH ₂—5); 5.46 (s, 2H, CH ₂—17) 7.01 (s, 1H,H-14); 7.40 (m, 2H, H-9+H-11); 7.99 (d, J=9.8 Hz, 1H, H—12); 8.33 (t,J=5.7 Hz. 1H, CONH—CH₂—CO).

EXAMPLE 6 Preparation of:7-ethyl-10-hydroxy-20-O-(6-aminohexanoyl-glycyl)camptothecin

hydrochloride

[6b: n=5, OCPT=(2d)]

7-ethyl-10-hydroxy-camptothecin (2d, 0.8 g, 2 mmol) was reacted withN-(tert-butoxycarbonyl)-6-aminohexanonyl-glycyl p-nitrophenylester (10a;2.5 g, 6 mmol) and 4-dimethyl amino pyridine as described in Example 5.The crude material, obtained from the reaction mixture by methylenechloride extraction, was dissolved in a mixture of 1.5N hydrochloricacid and acetic acid (20 ml). After standing for one hour under stirringat room temperature, the solution was reduced to small volume bydistillation and ethyl ether (100 ml) was added. The precipitate wascollected and washed with ethyl ether (2×50 ml) to give 1 g of the titlecompound (6b) as free amino derivative.

EXAMPLE 7 Preparation of MAG-camptothecin via Route I

[A1: n=5, OCPT=(2a)]

Polymeric precursor B (R₂=p-nitrophenyloxy, 2.58 g, containing 1.16×10³eq. of p-nitro phenyl ester residue), prepared as described inMakromol.Chem.178, 2159 (1977), was dissolved with dry dimethylsulfoxide(15ml) and added with 20-O-(6-aminohexanoyl-glycyl) camptothecintrifluoroacetate (6a; 0.63 g, 1 mmol), followed by triethylamine (0.14ml, 1 mmol). The reaction mixture was kept at room temperature for 22hours under stirring, then 2-propanolamine (0.05 ml) was added and themixture left under stirring for one more hour. After that, the reactionmixture was precipitated with ethyl acetate (200 ml) and left understirring for 30 min. The solid material was collected on a sinteredglass funnel, washed with ethyl acetate (200 ml) and ethyl ether (100ml) and then dissolved with ethanol (30 ml). The alcoholic solution wastreated with wet DOWEX-50, sulphonic form, (1.2 g) under stirring for 30min. and, after that, was added dropwise to n-hexane (200 ml). Theprecipitate was collected on a sintered glass funnel, washed with ethylether and dried to constant weight to give 2.68 g of the title compound(A1). Weight-average molecular weight (Mw): 19.800. Polydispersity(Mw/Mn): 1.5. Content of camptothecin, determined after alkalinehydrolysis, 10% w/w.

EXAMPLE 8 Preparation of MAG-(7-ethyl-10-hydroxycamptothecin) via RouteI

[A2: n=5, OCPT=(2d)]

Polymeric precursor (B) (R₂=p-nitrophenyloxy, 2.58 g, containing1.6×10⁻³ eq. of p-nitro phenyl ester residue), prepared as described inMakromol.Chem.178, 2159 (1977), was dissolved with dry dimethylsulfoxide(15 ml) and added with7-ethyl-10-hydroxy-20-O-(6-aminohexanoyl-glycyl)camptothecin trifluoroacetate (6b; 0.68 g. 1 mmol), followed by triethylamine (0.14 ml, 1mmol). The reaction mixture was kept at room temperature for 22 hoursunder stirring, then 2-propanolamine (0.05 ml) was added and the mixtureleft under stirring for one more hour. After that, the reaction mixturewas precipitated with ethyl acetate (200 ml) and left under stirring for30 min. The solid material was collected on a sintered glass funnel,washed with ethyl acetate (200 ml) and ethyl ether (100 ml) and thendissolved with ethanol (30 ml). The alcoholic solution was treated withwet DOWEX-50, sulphonic form, (1.2 g) under stirring for 30 min. and,after that, was added dropwise to n-hexane (200 ml). The precipitate wascollected on a sintered glass funnel, washed with ethyl ether and driedto constant weight to give 2.68 g of the title compound [A2]Weight-average molecular weight (Mw): 20.500.

Polydispersity (Mw/Mn): 1.87. Content of7-ethyl-10-hydroxy-camptothecin, determined after alkaline hydrolysis,10% w/w.

EXAMPLE 9 Preparation of MAG-camptothecin via Route II

[A1: n=5, OCPT=(2a)]

20-O-[methacryloyl-glycyl-(6-aminohexanoyl)-glycyl]camptothecin (9a:1.26g, 2 mmol), N-(2-hydroxypropyl)methacrylamide (8, 4.4 g, 31 mmol),prepared as described in Makromol. Chem.178, 2159 (1977), and2,2′-azobisisobutyronitrile (0.26 g, 1.6 mmol) were dissolved withanhydrous dimethysulfoxide (20 ml), kept at 60° C. under nitrogen andstirred for 24 hours. After that, the reaction mixture was cooled atroom temperature and poured into ethyl acetate (500 ml). The precipitateis collected and dissolved with ethanol (50 ml) from which isre-precipitated by adding ethyl acetate (500 ml). The solid iscollected, washed with ethyl acetate ethyl ether (2×100 ml) to give 5 gof the title compound (A1).

What is claimed is:
 1. A polymeric conjugate which consists of: (i) from85 to 97 mol % of N-(2-hydroxypropyl)methacryloylamide units representedby formula (3)

(ii) from 3 to 15 mol % of20-O-(N-methacryloyl-glycyl-aminoacyl-glycyl)camptothecin unitsrepresented by formula (4)

 wherein n is from 2 to 8, —[O-CPT] represents the residue of acamptothecin of formula (2)

 which is linked at the C-20 position and in which each of R₁, R₂, R₃,R₄ and R₅, which are the same or different, is hydrogen, C₁-C₁₂ linearor branched alkyl, nitro, amino, (CH₂)_(a)NR₆R₇ in which a is from 0 to4 and R₆ and R₇ are hydrogen or one of R₆ or R₇ is hydrogen and theother of R₆ or R₇ is C₁-C₆ alkyl, or NR₆R₇ represents a piperazino orN-alkyl-piperazino ring optionally substituted with C₁-C₆ linear orbranched alkyl, or a piperidino ring, (CH₂)_(a)NHCOR₈ in which a is asabove defined and R₈ is C₁-C₈ linear or branched alkyl or a group NR₆R₇as above, hydroxy or O—CO—R₈ in which R₈ is as above defined orrepresents a 1-piperidino ring or 1,4′-bipiperidine, or R₂ and R₃ takentogether represent the residue O—(CH₂)_(b)—O, in which b is 1 or 2, orR₄ and R₅ represent the residue (CH₂)_(m), in which m is from 2 to 4, orthe residue CH₂—O—CH₂ or CH₂NHCH₂ and (iii) from 0 to 12 mol % ofN-methacryloyl-glycine or N-(2-hydroxypropyl)methacryloyl-glycinamideunits represented by formula (5)

 wherein [Z] represents a hydroxy group or a residue of formula—NH—CH₂—CH(OH)—CH₃.
 2. A polymeric conjugate according to claim 1 whichcontains the N-(2-hydroxypropyl)methacryloylamide units represented bythe formula (3) in a molar proportion of 90%.
 3. A polymeric conjugateaccording to claim 1 which contains 10 mol % of the units represented bythe formula (4).
 4. A polymeric conjugate according to claim 1 whereinthe unit of formula (5) is absent.
 5. A polymeric conjugate according toclaim 1 in which —[O-CPT] in formula (4) is a residue of a camptothecinof formula (2) selected from: camptothecin, 9-aminocamptothecin,9-nitrocamptothecin, 7-ethyl-10-hydroxy-camptothecin,7-ethyl-10-[1,4′-bipiperidinyl]carbonyloxycamptothecin,7-methylendimethylamino-10-hydroxycamptothecin and7-[methylene-(4′-methylpiperazino)]-9,10-ethylendioxycamptothecin.
 6. Apolymeric conjugate according to claim 1 in which the content of activecamptothecin derivative of formula (2) is 10% (w/w).
 7. A process forproducing a polymeric conjugate as defined in claim 1, which processcomprises reacting a 20-O-(aminoacyl-glycyl)camptothecin derivative offormula (6) NH₂—(CH₂)_(n)—CO-Gly-[OCPT]  (6) wherein n and [O-CPT] areas defined in claim 1, with a polymer (B) consisting essentially of:from 85 to 97 mol % of N-(2-hydroxypropyl)methacryloylamide unitsrepresented by formula (3) as defined in claim 1, and from 3 to 15 mol %of N-methacryloyl-glycyl derivative units represented by formula (7)

wherein [Y] is the residue of an active ester or a hydroxy group; andoptionally displacing the remaining active ester groups with1-amino-2-propanol.
 8. A process for producing a polymeric conjugate asdefined in claim 1, which process comprises the polymerization betweenN-(2-hydroxypropyl)methacrylamide of the formula (8)

and 20-O-[methacryloyl-glycyl-(aminoacyl)-glycyl]camptothecinderivatives of the formula (9)

wherein n and [OCPT] are as defined in claim 1, in conditions capable ofpreserving the nature of linkage between camptothecin and spacerglycyl-aminoacyl-glycyl as well as that of the conjugate.
 9. A20-O-acylamino-glycyl-camptothecin derivative of the formula (6) asdefined in claim 7 or a salt thereof.
 10. A process for producing acompound of the formula (6) as defined in claim 7, which processcomprises condensing a derivative of formula (2) as defined above with aN-protected aminoacyl-glycyl derivative of formula (10):R₉—HN—(CH₂)_(n)—CO-Gly-[P]  (10) wherein n is as defined above, R₉represents an amino-protecting group and [P] is a residue of anactivated ester, to give a compound represented by formula (11):R₉—NH—(CH₂)_(n)—CO-Gly-[OCPT]  (11) wherein n and R₉ are as definedabove and [OCPT] is as defined above; and removing the N-protectinggroup from the resulting compound.
 11. A20-O-[methacryloyl-glycyl-(aminoacyl)-glycyl]camptothecin derivative ofthe formula (9) as defined in claim 8 or a salt thereof.
 12. A processfor producing a derivative of the formula (9) as defined in claim 11which process comprises condensing a camptothecin derivative of theformula (6) as defined above with N-methacryloyl-glycyl of formula (7′),

wherein [Y′] is a leaving group.
 13. A pharmaceutical compositioncomprising a pharmaceutically acceptable diluent or carrier and, asactive ingredient, a polymeric conjugate as defined in claim
 1. 14. Apolymeric conjugate according to claim 1 which is MAG-camptothecin, inwhich the camptothecin content is 10% (w/w).
 15. A polymeric conjugateaccording to claim 1 which is obtainable by reacting a20-O-(6-aminohexanoyl-glycyl)camptothecin derivative of formula (6)NH₂—(CH₂)₆—CO—Gly—[OCPT]  (6) wherein [O-CPT] is the residue ofcamptothecin, with a polymer (B) consisting essentially of: from 85 to97 mol % of N-(2-hydroxypropyl)methacryloylamide units represented byformula (3) as defined in claim 1, and from 3 to 15 mol % ofN-methacryloyl-glycyl derivative units represented by formula (7)

wherein [Y] is p-nitrophenoxy group; and optionally displacing theremaining active ester groups with 1-amino-2-propanol.
 16. A polymericconjugate according to claim 1 which is obtainable by the polymerizationbetween N-(2-hydroxypropyl)methacrylamide of the formula (8)

and 20-O-[methacryloyl-glycyl-(6-aminohexanoyl)-glycyl]camptothecinderivatives of the formula (9)

wherein [OCPT] is the residue of camptothecin.
 17. A pharmaceuticalcomposition comprising a pharmaceutically acceptable diluent or carrierand, as active ingredient, a polymeric conjugate as defined in claim 1.